In article <Pine.A126.96.36.1991010155459.136640D-100000 at spider.usp.br>,
scamargo at usp.br (Solange S de Camargo) wrote:
>>> Hi Colleagues,
>> I have to express a specific gene in a strain without selection
> marker. So, I'd like to know if it is easy to obtain lys - mutants and
> how I can do for get them. In addition, I'd like to know if somebody has a
> cloning vector for S. cerevisiae with the LYS2 gene as marker or the LYS2
> gene flanqued by a polilynker.
> I'd appreciate very much if somebody could help me.
> Best wishes,
Is there some reason why you cannot simply cross with a LYS- strain to
obtain waht you want? If you must make an isogenic LYS- derivative of the
strain you are presently using the best way would be to engineer a LYS
disruption on a plasmid and transform your strain with the disrupted LYS
gene fragment (and e.g. a small amount of episomal plasmid as a secondary
marker to select for the yeast that took up DNA -- in most cases you will
be able to cure the episomal plasmid afterwards)
I am trying to remember if LYS is one of the genes that you can select
against --in the back of my mind I remember that it is -- I think either
Current Protocols or one of the yeast technique manuals e.g. (Fink) give
details. If this is so then you can simply transform with your disrupted
LYS gene fragment (without a secondary marker) and use a negative selection
technique to obtain LYS- mutants.